Biology subjects

Gouet, P.

Publications and source records attributed to Gouet, P..

4 recordsLinked to original sources

bioRxiv2024

A nanobody-based approach to capture and visualize interactions of binary protein complexes in living cells

Protein interaction networks (or interactomes) are formed progressively, each interaction influencing the next one. Accordingly, a same protein will establish different interactomes depending on its first associated cofactor, thereby diversifying its function in the cell. In contrast to their central role, few methods exist to capture interactomes of dimeric protein complexes. Here, we tackle this issue by introducing an innovative method based on bimolecular fluorescence complementation and the specific binding of a nanobody fused to a proximity-dependent biotinylating enzyme. This method was applied to visualize and capture specific interactions of the cytoplasmic TAZ/14-3-3e and nuclear TAZ/TEAD2 complexes, which are major downstream effectors of the Hippo signaling pathway. Among other interactions, we identified SERPINB4 as a novel regulator of TAZ and 14-3-3e proliferative activity in mesenchymal stromal cells. Molecular dissections in living cells revealed the central role of a unique residue of TAZ for recruiting SERPINB4 specifically in the presence of 14-3-3e. Overall, our work demonstrates the importance of considering binary protein complexes for deciphering interactomes and establishes a novel sensitive method in this perspective.

molecular biology↗

bioRxiv2024

The SARS-CoV-2 nucleocapsid protein inhibits the cellular Nonsense-Mediated mRNA Decay (NMD) pathway preventing the full enzymatic activation of UPF1.

The Nonsense-mediated mRNA decay (NMD) pathway triggers the degradation of defective mRNAs and governs the expression of mRNAs with specific characteristics. Current understanding indicates that NMD is often significantly suppressed during viral infections to protect the viral genome. In numerous viruses, this inhibition is achieved through direct or indirect interference with the RNA helicase UPF1, thereby promoting viral replication and enhancing pathogenesis. In this study, we employed biochemical, biophysical assays, and cellular investigations to explore the interplay between UPF1 and the Nucleocapsid (Np) protein of SARS-CoV-2. We evaluated their direct interaction and its impact on inhibiting cellular NMD. Furthermore, we characterized how this interaction affects UPF1s enzymatic function. Our findings demonstrate that Np inhibits the unwinding activity of UPF1 by physically obstructing its access to structured nucleic acid substrates. Additionally, we showed that Np binds directly to UPF2, disrupting the formation of the UPF1/UPF2 complex essential for NMD progression. Intriguingly, our research also uncovered a surprising pro-viral role of UPF1 and an antiviral function of UPF2. These results unveil a novel, multi-faceted mechanism by which SARS-CoV-2 evades the hosts defenses and manipulates cellular components. This underscores the potential therapeutic strategy of targeting Np-UPF1/UPF2 interactions to treat COVID-19.

biochemistry↗

bioRxiv2021

Collinearity of alpha-helices or beta strands in membrane proteins causes a characteristic peak centred on 4.9 A resolution in diffraction intensity profiles, inducing higher diffraction anisotropy

Diffraction anisotropy is a phenomenon that impacts more specifically membrane proteins, compared to soluble ones, but the reasons for this discrepancy remained unclear. Often, it is referred to a difference in resolution limits between highest and lowest diffraction limits as a signature for anisotropy. We show in this article that there is no simple correlation between anisotropy and difference in resolution limits, with notably a substantial number of structures displaying various anisotropy with no difference in resolution limits. We further investigated diffraction intensity profiles, and observed a peak centred on 4.9[A] resolution more predominant in membrane proteins. Since this peak is in the region corresponding to secondary structures, we investigated the influence of secondary structure ratio. We showed that secondary structure content has little influence on this profile, while secondary structure collinearity in membrane proteins correlate with a stronger peak. Finally, we could further show that the presence of this peak is linked to higher diffraction anisotropy. SynopsisMembrane protein diffraction anisotropy originates from a peak at 4.9 [A] resolution in intensity profiles, due to secondary structure collinearity.

biophysics↗

bioRxiv2021

The HIV-1 Integrase C-Terminal domain induces TAR RNA structural changes promoting Tat binding.

Recent evidence indicated that HIV-1 Integrase (IN) binds genomic viral RNA (gRNA) playing a critical role in viral particle morphogenesis and gRNA stability in host cells. Combining biophysical and biochemical approaches we show that the C-terminal flexible 18-residues tail of IN acts as a sensor of the peculiar apical structure of trans-activation response element RNA (TAR), directly interacting with its hexaloop. We highlighted how the whole IN C-terminal domain, once bound to TAR, can change its structure assisting the binding of Tat, the HIV trans-activator protein, which finally displaces IN from TAR. Our results are consistent with the emerging role of IN in early stage of proviral transcription and suggest new steps of HIV-1 life cycle that can be considered as therapeutic targets.

biochemistry↗